DNA fragment comprising a gene encoding ethylene forming enzyme of bacteria and the use thereof

ABSTRACT

PCT No. PCT/JP93/01309 Sec. 371 Date Mar. 1, 1994 Sec. 102(e) Date Mar. 1, 1994 PCT Filed Sep. 14, 1993 PCT Pub. No. WO94/06914 PCT Pub. Date Mar. 31, 1994.Bacteria such as E. coli which are easy in manipulation are transformed by the gene encoding a ethylene-forming enzyme, and ethylene is formed by the transformants.

TECHNICAL FIELD

This invention relates to a DNA fragment comprising a gene encoding anethylene-forming enzyme of bacteria, a vector including the DNAfragment, transformants transformed with the vector, and a method forproducing ethylene using the transformants.

BACKGROUND ART

While ethylene is produced from crude petroleum or natural gas, it haslong been known that ethylene could also be formed by plants andmicroorganisms.

Chou et al. reported that α-ketoglutaric acid(α-KG) or L-glutamicacid(Glu) is a precursor of ethylene in Penicillium digitatum, a speciesof fungi (Arch. Biochem. Biophys., 157, 73, 1973), and Goto et al.reported that α-KG serves as a substrate in the ethylene forming systemin cell-free extracts of Pseudomonas syringae which is one of thespecies of pathogenic bacteria for plants (Plant Cell Physiol., 28, 405,1987).

Primrose et al. reported that, in the ethylene-forming system incell-free extracts of Escherichia coli, 2-keto-4-methylthiobutyricacid(KMBA) which is a metabolic intermediate of L-methionine(Met) is aprecursor of ethylene biosynthesis (J. Gen. Microbiol.,98,519, 1977). Inall these reports, however, the true substrates and biosynthetic pathwayof ethylene-forming reaction were not established because materials suchas cultured cells of bacteria or their cell free extracts, which aresupposed to contain a lot of impurities, were used in the experiments.

To make clear the pathway of the ethylene biosynthesis caused by thebacteria and the ethylene-forming enzyme reaction, the present inventorspurified the enzyme catalyzing the ethylene formation toelectrophoretically homogeneous state. The results revealed that theethylene-forming reaction via KMBA is really radical reactions in whichactive oxygen is concerned (Fukuda et al., FEMS Microbiol. Lett., 60,107, 1989). We have also investigated the ethylene-forming enzyme ofPenicillium digitatum IFO 9372 via a α-KG and its enzymatic reaction(Fukuda et al., FEMS Microbiol.lett., 59, 1989), and theethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 viaα-KG and its characteristics (Nagahama, Fukuda et al., J. Gen.Microbiol., 137, 2228, 1991).

Although the enzyme catalyzing the ethylene biosynthesis by bacteria wasidentified as well as the characteristics of the enzyme, by the presentinventors as described above, the ability of ethylene formation in theseethylene-forming bacteria was not adequately sufficient.

For the purpose of fundamental breeding improvement of theethylene-forming bacteria through a gene manipulation technique,Pseudomonas syringae pv. phaseolicola PK2 was selected for the objectand its DNA sequence encoding the ethylene-forming enzyme was analyzed,thereby completing this invention.

DISCLOSURE OF INVENTION

To achieve the above described object, this invention is characterizedin that a gene encoding an amino acid sequence represented by:

Sea ID No: 1 Met Thr Asn Leu Gln Thr Phe Glu Leu Pro Thr Glu Val Thr GlyCys Ala Ala Asp Ile Ser Leu Gly Arg Ala Leu Ile Gln Ala Trp Gln Lys AspGly Ile Phe Gln Ile Lys Thr Asp Ser Glu Gln Asp Arg Lys Thr Gln Glu AlaMet Ala Ala Ser Lys Gln Phe Cys Lys Glu Pro Leu Thr Phe Lys Ser Ser CysVal Ser Asp Leu Thr Tyr Ser Gly Tyr Val Ala Ser Gly Glu Glu Val Thr AlaGly Lys Pro Asp Phe Pro Glu Ile Phe Thr Val Cys Lys Asp Leu Ser Val GlyAsp Gln Arg Val Lys Ala Gly Trp Pro Cys His Gly Pro Val Pro Trp Pro AsnAsn Thr Tyr Gln Lys Ser Met Lys Thr Phe Met Glu Glu Leu Gly Leu Ala GlyGlu Arg Leu Leu Lys Leu Thr Ala Leu Gly Phe Glu Leu Pro Ile Asn Thr PheThr Asp Leu Thr Arg Asp Gly Trp His His Met Arg Val Leu Arg Phe Pro ProGln Thr Ser Thr Leu Ser Arg Gly Ile Gly Ala His Thr Asp Tyr Gly Leu LeuVal Ile Ala Ala Gln Asp Asp Val Gly Gly Leu Tyr Ile Arg Pro Pro Val GluGly Glu Lys Arg Asn Arg Asn Trp Leu Pro Gly Glu Ser Ser Ala Gly Met PheGlu His Asp Glu Pro Trp Thr Phe Val Thr Pro Tbr Pro Gly Val Trp Tbr ValPhe Pro Gly Asp Ile Leu Gln Phe Met Thr Gly Gly Gln Leu Leu Ser Thr ProHis Lys Val Lys Leu Asn Thr Arg Glu Arg Phe Ala Cys Ala Tyr Phe His GluPro Asn Phe Glu Ala Ser Ala Tyr Pro Leu Phe Glu Pro Ser Ala Asn Glu ArgIle His Tyr Gly Glu His Phe Thr Asn Met Phe Met Arg Cys Tyr Pro Asp ArgIle Thr Thr Gln Arg Ile Asn Lys Glu Asn Arg Leu Ala His Leu Glu Asp LeuLys Lys Tyr Ser Asp Thr Arg Ala Thr Gly Ser

is integrated into bacteria.

DNA encoding the ethylene-forming enzyme of the present invention can beobtained, for example, by the procedures comprising:

preparing a probe with an amino acid sequence coding theethylene-forming enzyme from Pseudomonas syringae; hybridizing thisprobe with an indigenous plasmid by Southern method to find that a genefor the ethylene-forming enzyme is encoded in the indigenous plasmidDNA; constructing a gene library after transforming Escherichia coli (E.coli) using restriction enzyme Hind III fragments of this indigenousplasmid DNA and pUC19 as a vector; and screening a positive clone E.coli JM109 (pEFE01) from the library by a hybridization using Southernmethod.

Hind III fragments of about 2.5 kbp from Pseudononas syringae wereinserted into the plasmid pUC19 in the cell of this positive clone E.coli JMO109(pEFE01), and an ethylene-forming activity was found in thispositive clone E. coli JM109(pEFE01). This positive clone E. coliJM109(pEFE01) also expressed a protein that could not be distinguishedfrom the ethylene-forming enzyme protein of Pseudomonas syringae asdetected by Western blotting method using an antibody for the ethyleneforming enzyme.

Deletion mutant strains having various sizes of plasmids were preparedby digesting the inserted 2.5kbp Hind III fragments. The smallest pEFEO1derivative was obtained from the mutants keeping ethylene-formingactivity. The size of the smallest fragment was about 1.5 kbp.

BRIEF DESCRIPTION OF DRAWOMGS

FIG. 1 shows a base sequence of the gene of the ethylene-forming enzymeafter cloning and the first half portion of the amino acid sequencetranslated therefrom.

FIG. 1 shows a base sequence of the gene of the ethylene-forming enzymeafter cloning and the latter half portion of the amino acid sequencetranslated therefrom.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLES

Chromosomal DNA of Pseudomonas syringae was extracted with phenol afterlysis of cell wall with lysozyme and sodium dodecyl gulfate (SDS),wherein proteins were removed by denaturation and DNA was recovered fromthe supernatant by ethanol precipitation. The plasmid DNA of Pseudomonassyringae wae was extracted by an alkali extraction method.

Extracted DNA was digested with a restriction enzyme Hind III and thedigested fragments were subjected to electrophoresis using 0.7% ofagarose gel. DNA in the gel after the electrophoresis were transferredto a Nylon membrane by a capillary method, bound onto the membrane by adry-heat treatment and were subjected to Southern hybridization.

Since the structural gene of the ethylene-forming enzyme (EFE) seemed tobe located on the plasmid after the hybridization, the plasmid DNAfragments digested with Hind III are ligated with the E. coli vector(pUC19) subjected to an alkali phosphatase treatment, thereby obtainingthe chimera plasmid. This chimera plasmid was mixed with competent cellsof Escherichia coli (E. coli YM109) prepared by a calcium chloridemethod and, after allowing them to stand in an ice bath for an hour, thechimera plasmid was incorporated into the cell with a heat shock,followed by centrifugation, thereby transforming E. coli JM108. This E.coli JM109 after the transformation was seeded in 2xYT culture mediumconsisting of 16g/liter of Bactotrypton, 10g/liter of Bactoyeast extractand 5g/liter of sodium chloride. After the cultivation for 1.5 hours, E.coii JM109 was separated by centrifugation and inoculated in a selectedculture medium.

Colonies having the chimera plasmid expressed in the selected culturemedium were screened and these colonies were cultivated in LB culturemedium consisting of 10g/liter of Bactotrypton, 5g/liter of Bactoyeastextract and 10g/liter of sodium chloride supplemented with 5 μg/ml ofthiamine hydrochloride and 50 μg/ml of ampicillin sodium salt. Theplasmid DNA (chimera pUC19) was extracted with an aqueous alkaline,purified by removing RNA and, after treating with Hind III, it wassubjected to electrophoresis using 1% of agarose gel. The DNA afterelectrophoresis was denatured with an aqueous alkaline solution followedby neutralization and blotted on a nylon membrane, and then E. coliwhich retained the gene of the ethylene-forming enzyme was screened byhybridization.

When E. coli JM109(pEFE01) was cultivated in the above described LBculture medium supplemented with 5g/ml of L-glutamic acid, 5 μg/ml ofthiamine hydrochloride and 50 μg/ml of ampicillin sodium salt, theethylene-forming activity was 230nl/ml of culture medium/hr. In theabove described E. coli JM109 (a disclosed strain well known in the art)used as a host, on the other hand, any ethylene-forming activity was notdetected and its ethylene-forming rate was On1/ml of culture medium/hr.The term "ethylene-forming activity" used herein refers to an in vitroethylene-forming ability in a cell-free extract prepared from the cells,and the term "ethylene-forming rate" refers to an in vivoethylene-forming ability in the culture medium. These characteristicscould be detected by conventional methods described, for example, in theabove described reference (Nagahama, Fukuda et al., J. Gen. Microbiol.,37, 2281, 1991).

The transformant Escherichia coli JM109(pEFE01) used in this inventionwas deposited to National Institute of Bioscience and Human-Technology,1-3, Higashi -chome, Tsukuba City, Japan, under the registration numberof FERM P-1361 dated Sep. 16, 1992.

It was made clear by Western blotting that an identical protein with theethylene-forming enzyme of Pseudomonas syringae was also present in E.coli JM109(pEFE01). Deletion mutant having various size of plasmids wereprepared by digesting the inserted 2.5kbp Hind III fragment from itsterminal. The smallest pEFE01 transformant retaining theethylene-forming activity was about 1.5 kbp. In addition, neither sitefor Hind III nor sites for other restriction enzymes PstI, Dra I, EcoR Iand BamH I were present in this fragment.

The base sequence and amino acid sequence of the gene of the ethyleneforming-enzyme incorporated in 2.5 kbp Hind III fragment as determinedby a dideoxy method are shown in FIG. 1 and FIG. 2.

Industrial Applicability

According to this invention, bacteria having high ethylene-formingactivity can be obtained by transforming bacteria such as E. coli whichis safe and easy in manipulation.

Moreover, ethylene can be produced with high efficiency by utilizingthese transformed bacteria.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 350 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetThrAsnLeuGlnThrPheGluLeuProThrGluValThrGlyCys                              151015                                                                        AlaAlaAspIleSerLeuGlyArgAlaLeuIleGlnAlaT rpGlnLys                             202530                                                                        AspGlyIlePheGlnIleLysThrAspSerGluGlnAspArgLysThr                              3540 45                                                                       GlnGluAlaMetAlaAlaSerLysGlnPheCysLysGluProLeuThr                              505560                                                                        PheLysSerSerCysValSerAspLeuThrTyrSerGlyTyrValAla                              65707580                                                                      SerGlyGluGluValThrAlaGlyLysProAspPheProGluIlePhe                              8590 95                                                                       ThrValCysLysAspLeuSerValGlyAspGlnArgValLysAlaGly                              100105110                                                                     TrpProCysHisGlyProValProTrpProAsnAsn ThrTyrGlnLys                             115120125                                                                     SerMetLysThrPheMetGluGluLeuGlyLeuAlaGlyGluArgLeu                              13013514 0                                                                    LeuLysLeuThrAlaLeuGlyPheGluLeuProIleAsnThrPheThr                              145150155160                                                                  AspLeuThrArgAspGlyTrpHisHisMetArgVal LeuArgPhePro                             165170175                                                                     ProGlnThrSerThrLeuSerArgGlyIleGlyAlaHisThrAspTyr                              180185 190                                                                    GlyLeuLeuValIleAlaAlaGlnAspAspValGlyGlyLeuTyrIle                              195200205                                                                     ArgProProValGluGlyGluLysArgAsnAr gAsnTrpLeuProGly                             210215220                                                                     GluSerSerAlaGlyMetPheGluHisAspGluProTrpThrPheVal                              225230235 240                                                                 ThrProThrProGlyValTrpThrValPheProGlyAspIleLeuGln                              245250255                                                                     PheMetThrGlyGlyGlnLeuLeuS erThrProHisLysValLysLeu                             260265270                                                                     AsnThrArgGluArgPheAlaCysAlaTyrPheHisGluProAsnPhe                              275 280285                                                                    GluAlaSerAlaTyrProLeuPheGluProSerAlaAsnGluArgIle                              290295300                                                                     HisTyrGlyGluHisPheThrAsnMetPhe MetArgCysTyrProAsp                             305310315320                                                                  ArgIleThrThrGlnArgIleAsnLysGluAsnArgLeuAlaHisLeu                              325 330335                                                                    GluAspLeuLysLysTyrSerAspThrArgAlaThrGlySer                                    340345350                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1050 base pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATGACCAACCTACAGACTTTCGAGTTGCCTACCGAGGTAACCGGCTGCGCCGCCGATATC60                TCATTGGGAAGGGCGCTGATCCA AGCCTGGCAAAAAGATGGCATTTTTCAGATCAAGACC120              GATAGTGAGCAGGATCGCAAAACGCAGGAAGCAATGGCTGCTAGCAAGCAGTTTTGCAAG180               GAACCGCTGACTTTTAAGAGTAGCTGCGTTAGCGATCTGACCTACAGCGGCTATGTTGCG240               TCAGGCGAGGAAGTCACAGCTGGTAAACCTGATTTCCCTGAAATCTTCACTGTCTGCAAG300               GACTTGTCGGTAGGCGATCAGCGTGTAAAAGCCGGCTGGCCTTGCCATGGTCCGGTGCCA360               TGGCCAAATAACACCTATCAGAAAAGCATGAAGACCTTCAT GGAAGAGCTGGGTTTAGCG420              GGCGAACGGTTGCTCAAACTGACAGCGCTCGGCTTTGAACTACCCATCAACACGTTCACC480               GACTTAACTCGCGATGGTTGGCACCACATGCGTGTATTACGCTTCCCGCCCCAAACATCC540               ACGCTGTCCCGTGGAA TTGGTGCGCACACTGACTATGGGTTGTTGGTAATTGCCGCTCAG600              GACGATGTTGGTGGCTTATATATTCGCCCTCCAGTCGAGGGAGAGAAGCGTAATCGTAAC660               TGGTTGCCTGGTGAGAGCTCAGCAGGCATGTTTGAGCACGATGAACCTTGGACCTTCGTG 720              ACGCCCACCCCAGGCGTGTGGACAGTTTTCCCAGGTGATATCTTGCAGTTCATGACCGGC780               GGCCAGCTGCTTTCCACTCCGCACAAGGTTAAGCTCAATACCCGCGAACGTTTCGCCTGC840               GCTTATTTTCATGAGCCTAATTTTGAAGCATCCG CCTATCCGTTGTTCGAGCCCAGCGCC900              AATGAGCGTATTCATTATGGTGAGCACTTTACCAACATGTTTATGCGTTGCTATCCAGAT960               CGGATCACCACCCAGAGGATCAACAAGGAGAATCGCCTGGCGCACTTGGAGGACTTGAAG1020              AAGTATTCG GACACCCGCGCGACAGGCTCA1050                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1809 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGACCGCGTGTCAGCAACGACAATCCTTACTCGGAGTCGCTGTTCAGGACACTGAAGTA60                CTGCCCGCAATGGCCGCAGGATGGGTTTGCCAGTCTTGACGCGGCACGCTCTGGGTGAGG120               GATTTCATGCGTTGGTACAACAATGATCACCGGCACAGCCGA ATCCGCTTCGTGACACCG180              GCTGAGCGGCATCGAGGGCTGGATCATCAGATCCTGGCCAAGCGACATGAGCTGTACGAG240               CTAGCCAGAGAGAAAAGGCCGGAGCGGTGGTCGAGGGAGACACGCAACTGGGAACCGATC300               GGCACCGTGCTGTTAAA CCCGGATCGAGAGCAAGACGTTGAGAAAAAAGCAGCATAGTTA360              GACGGTTGACGCGACAACTACCTTGAAAAACGCCGGCAGGGACGCTCATGATTCATGCTC420               CAAGCAGGTGGGGTGTTTTTCCGTCGCTTGGACTTTGTTCTCCCGACGTCGTTTGGAATG 480              AGCATCCGTCCCTTTATATGGATAAAGAAGAGACTAGCATGACCAACCTACAGACTTTCG540               AGTTGCCTACCGAGGTAACCGGCTGCGCCGCCGATATCTCATTGGGAAGGGCGCTGATCC600               AAGCCTGGCAAAAAGATGGCATTTTTCAGATCAAG ACCGATAGTGAGCAGGATCGCAAAA660              CGCAGGAAGCAATGGCTGCTAGCAAGCAGTTTTGCAAGGAACCGCTGACTTTTAAGAGTA720               GCTGCGTTAGCGATCTGACCTACAGCGGCTATGTTGCGTCAGGCGAGGAAGTCACAGCTG780               GTAAACCTGA TTTCCCTGAAATCTTCACTGTCTGCAAGGACTTGTCGGTAGGCGATCAGC840              GTGTAAAAGCCGGCTGGCCTTGCCATGGTCCGGTGCCATGGCCAAATAACACCTATCAGA900               AAAGCATGAAGACCTTCATGGAAGAGCTGGGTTTAGCGGGCGAACGGTTGCTC AAACTGA960              CAGCGCTCGGCTTTGAACTACCCATCAACACGTTCACCGACTTAACTCGCGATGGTTGGC1020              ACCACATGCGTGTATTACGCTTCCCGCCCCAAACATCCACGCTGTCCCGTGGAATTGGTG1080              CGCACACTGACTATGGGTTGTTGGTAAT TGCCGCTCAGGACGATGTTGGTGGCTTATATA1140             TTCGCCCTCCAGTCGAGGGAGAGAAGCGTAATCGTAACTGGTTGCCTGGTGAGAGCTCAG1200              CAGGCATGTTTGAGCACGATGAACCTTGGACCTTCGTGACGCCCACCCCAGGCGTGTGGA1260              CA GTTTTCCCAGGTGATATCTTGCAGTTCATGACCGGCGGCCAGCTGCTTTCCACTCCGC1320             ACAAGGTTAAGCTCAATACCCGCGAACGTTTCGCCTGCGCTTATTTTCATGAGCCTAATT1380              TTGAAGCATCCGCCTATCCGTTGTTCGAGCCCAGCGCCAATGAGCG TATTCATTATGGTG1440             AGCACTTTACCAACATGTTTATGCGTTGCTATCCAGATCGGATCACCACCCAGAGGATCA1500              ACAAGGAGAATCGCCTGGCGCACTTGGAGGACTTGAAGAAGTATTCGGACACCCGCGCGA1560              CAGGCTCATGAGTCGACACC CTGCCCGGTGCTGCCGGACAGGGGCCTTATCGTTACTGGT1620             GACTAATAATTGGCATATCAATGTCCACTCAGCACCCAGATCTTATGATCTGGGTGCTGA1680              GTGGAGCAATGTAACCATTATGCTGAGCGTTCATGCATAGGAATTTCAATAATTCCTATA17 40             CAAGGCAATCCGCCGAAAAAGGTCCCCTCGGCGTGATGCCAACGTGGCGTCGATGTCGGC1800              AAAAAGCTT1809                                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1809 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 519..1568                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCGACCGCGTGTCAGCAACGACAATCCTTACTCGGAGTCGCTGTTCAGGACAC TGAAGTA60               CTGCCCGCAATGGCCGCAGGATGGGTTTGCCAGTCTTGACGCGGCACGCTCTGGGTGAGG120               GATTTCATGCGTTGGTACAACAATGATCACCGGCACAGCCGAATCCGCTTCGTGACACCG180               GCTGAGCGGCATCGAGGGCTGGATCATC AGATCCTGGCCAAGCGACATGAGCTGTACGAG240              CTAGCCAGAGAGAAAAGGCCGGAGCGGTGGTCGAGGGAGACACGCAACTGGGAACCGATC300               GGCACCGTGCTGTTAAACCCGGATCGAGAGCAAGACGTTGAGAAAAAAGCAGCATAGTTA360               GA CGGTTGACGCGACAACTACCTTGAAAAACGCCGGCAGGGACGCTCATGATTCATGCTC420              CAAGCAGGTGGGGTGTTTTTCCGTCGCTTGGACTTTGTTCTCCCGACGTCGTTTGGAATG480               AGCATCCGTCCCTTTATATGGATAAAGAAGAGACTAGCATGACC AACCTACAG533                     MetThrAsnLeuGln                                                               15                                                                            ACTTTCGAGTTGCCTACCGAGGTAACCGGCTGCGC CGCCGATATCTCA581                          ThrPheGluLeuProThrGluValThrGlyCysAlaAlaAspIleSer                              101520                                                                        TTGGGAAGGGCGCTGATCCAAGCCTGGCAAAA AGATGGCATTTTTCAG629                          LeuGlyArgAlaLeuIleGlnAlaTrpGlnLysAspGlyIlePheGln                              253035                                                                        ATCAAGACCGATAGTGAGCAGGATCGCAAAAC GCAGGAAGCAATGGCT677                          IleLysThrAspSerGluGlnAspArgLysThrGlnGluAlaMetAla                              404550                                                                        GCTAGCAAGCAGTTTTGCAAGGAACCGCTGACTTT TAAGAGTAGCTGC725                          AlaSerLysGlnPheCysLysGluProLeuThrPheLysSerSerCys                              556065                                                                        GTTAGCGATCTGACCTACAGCGGCTATGTTGCGTCAGGCGA GGAAGTC773                          ValSerAspLeuThrTyrSerGlyTyrValAlaSerGlyGluGluVal                              70758085                                                                      ACAGCTGGTAAACCTGATTTCCCTGAAATCTTCAC TGTCTGCAAGGAC821                          ThrAlaGlyLysProAspPheProGluIlePheThrValCysLysAsp                              9095100                                                                       TTGTCGGTAGGCGATCAGCGTGTAAAAGCCGG CTGGCCTTGCCATGGT869                          LeuSerValGlyAspGlnArgValLysAlaGlyTrpProCysHisGly                              105110115                                                                     CCGGTGCCATGGCCAAATAACACCTATCAGAA AAGCATGAAGACCTTC917                          ProValProTrpProAsnAsnThrTyrGlnLysSerMetLysThrPhe                              120125130                                                                     ATGGAAGAGCTGGGTTTAGCGGGCGAACGGTTGCT CAAACTGACAGCG965                          MetGluGluLeuGlyLeuAlaGlyGluArgLeuLeuLysLeuThrAla                              135140145                                                                     CTCGGCTTTGAACTACCCATCAACACGTTCACCGACTTAAC TCGCGAT1013                         LeuGlyPheGluLeuProIleAsnThrPheThrAspLeuThrArgAsp                              150155160165                                                                  GGTTGGCACCACATGCGTGTATTACGCTTCCCGCC CCAAACATCCACG1061                         GlyTrpHisHisMetArgValLeuArgPheProProGlnThrSerThr                              170175180                                                                     CTGTCCCGTGGAATTGGTGCGCACACTGACTA TGGGTTGTTGGTAATT1109                         LeuSerArgGlyIleGlyAlaHisThrAspTyrGlyLeuLeuValIle                              185190195                                                                     GCCGCTCAGGACGATGTTGGTGGCTTATATAT TCGCCCTCCAGTCGAG1157                         AlaAlaGlnAspAspValGlyGlyLeuTyrIleArgProProValGlu                              200205210                                                                     GGAGAGAAGCGTAATCGTAACTGGTTGCCTGGTGA GAGCTCAGCAGGC1205                         GlyGluLysArgAsnArgAsnTrpLeuProGlyGluSerSerAlaGly                              215220225                                                                     ATGTTTGAGCACGATGAACCTTGGACCTTCGTGACGCCCAC CCCAGGC1253                         MetPheGluHisAspGluProTrpThrPheValThrProThrProGly                              230235240245                                                                  GTGTGGACAGTTTTCCCAGGTGATATCTTGCAGTT CATGACCGGCGGC1301                         ValTrpThrValPheProGlyAspIleLeuGlnPheMetThrGlyGly                              250255260                                                                     CAGCTGCTTTCCACTCCGCACAAGGTTAAGCT CAATACCCGCGAACGT1349                         GlnLeuLeuSerThrProHisLysValLysLeuAsnThrArgGluArg                              265270275                                                                     TTCGCCTGCGCTTATTTTCATGAGCCTAATTT TGAAGCATCCGCCTAT1397                         PheAlaCysAlaTyrPheHisGluProAsnPheGluAlaSerAlaTyr                              280285290                                                                     CCGTTGTTCGAGCCCAGCGCCAATGAGCGTATTCA TTATGGTGAGCAC1445                         ProLeuPheGluProSerAlaAsnGluArgIleHisTyrGlyGluHis                              295300305                                                                     TTTACCAACATGTTTATGCGTTGCTATCCAGATCGGATCAC CACCCAG1493                         PheThrAsnMetPheMetArgCysTyrProAspArgIleThrThrGln                              310315320325                                                                  AGGATCAACAAGGAGAATCGCCTGGCGCACTTGGA GGACTTGAAGAAG1541                         ArgIleAsnLysGluAsnArgLeuAlaHisLeuGluAspLeuLysLys                              330335340                                                                     TATTCGGACACCCGCGCGACAGGCTCATGAGTC GACACCCTGCCCGG1588                          TyrSerAspThrArgAlaThrGlySer                                                   345350                                                                        TGCTGCCGGACAGGGGCCTTATCGTTACTGGTGACTAATAATTGGCATATCAATGTCCAC1648              TCAGCACCCAGAT CTTATGATCTGGGTGCTGAGTGGAGCAATGTAACCATTATGCTGAGC1708             GTTCATGCATAGGAATTTCAATAATTCCTATACAAGGCAATCCGCCGAAAAAGGTCCCCT1768              CGGCGTGATGCCAACGTGGCGTCGATGTCGGCAAAAAGCTT 1809                                __________________________________________________________________________

What is claimed is:
 1. A DNA fragment comprising a gene encoding anethylene-forming enzyme of bacteria represented by the following aminoacid sequence:SEQ ID NO:1:Met Thr Asn Leu Gln Thr Phe Glu Leu Pro ThrGlu Val Thr Gly Cys Ala Ala Asp Ile Ser Leu Gly Arg Ala Leu Ile Gln AlaTrp Gln Lys Asp Gly Ile Phe Gln Ile Lys Thr Asp Ser Glu Gln Asp Arg LysThr Gln Glu Ala Met Ala Ala Ser Lys Gln Phe Cys Lys Glu Pro Leu Thr PheLys Ser Ser Cys Val Ser Asp Leu Thr Tyr Ser Gly Tyr Val Ala Ser Gly GluGlu Val Thr Ala Gly Lys Pro Asp Phe Pro Glu Ile Phe Thr Val Cys Lys AspLeu Ser Val Gly Asp Gln Arg Val Lys Ala Gly Trp Pro Cys His Gly Pro ValPro Trp Pro Asn Asn Thr Tyr Gln Lys Ser Met Lys Thr Phe Met Glu Glu LeuGly Leu Ala Gly Glu Arg Leu Leu Lys Leu Thr Ala Leu Gly Phe Glu Leu ProIle Asn Thr Phe Thr Asp Leu Thr Arg Asp Gly Trp His His Met Arg Val LeuArg Phe Pro Pro Gln Thr Ser Thr Leu Ser Arg Gly Ile Gly Ala His Thr AspTyr Gly Leu Leu Val Ile Ala Ala Gln Asp Asp Val Gly Gly Leu Tyr Ile ArgPro Pro Val Glu Gly Glu Lys Arg Asn Arg Asn Trp Leu Pro Gly Glu Ser SerAla Gly Met Phe Glu His Asp Glu Pro Trp Thr Phe Val Thr Pro Thr Pro GlyVal Trp Thr Val Phe Pro Gly Asp Ile Leu Gln Phe Met Thr Gly Gly Gln LeuLeu Ser Thr Pro His Lys Val Lys Leu Asn Thr Arg Glu Arg Phe Ala Cys AlaTyr Phe His Glu Pro Asn Phe Glu Ala Ser Ala Tyr Pro Leu Phe Glu Pro SerAla Asn Glu Arg Ile His Tyr Gly Glu His Phe Thr Asn Met Phe Met Arg CysTyr Pro Asp Arg Ile Thr Thr Gln Arg Ile Asn Lys Glu Asn Arg Leu Ala HisLeu Glu Asp Leu Lys Lys Tyr Ser Asp Thr Arg Ala Thr Gly Ser
 2. A DNAfragment according to claim 1 which comprises a gene encoding anethylene-forming enzyme of bacteria, wherein said bacteria belong to agenus Pseudomonas.
 3. A DNA fragment according to claim 1 whichcomprises a gene encoding an ethylene-forming enzyme of bacteriarepresented by the following base sequence:SEQ ID NO:2ATG ACC AAC CTACAG ACT TTC GAG TTG CCT ACC GAG GTA ACC GGC TGC GCC GCC GAT ATC TCA TTGGGA AGG GCG CTG ATC CAA GCC TGG CAA AAA GAT GGC ATT TTT CAG ATC AAG ACCGAT AGT GAG CAG GAT CGC AAA ACG CAG GAA GCA ATG GCT GCT AGC AAG CAG TTTTGC AAG GAA CCG CTG ACT TTT AAG AGT AGC TGC GTT AGC GAT CTG ACC TAC AGCGGC TAT GTT GCG TCA GGC GAG GAA GTC ACA GCT GGT AAA CCT GAT TTC CCT GAAATC TTC ACT GTC TGC AAG GAC TTG TCG GTA GGC GAT CAG CGT GTA AAA GCC GGCTGG CCT TGC CAT GGT CCG GTG CCA TGG CCA AAT AAC ACC TAT CAG AAA AGC ATGAAG ACC TTC ATG GAA GAG CTG GGT TTA GCG GGC GAA CGG TTG CTC AAA CTG ACAGCG CTC GGC TTT GAA CTA CCC ATC AAC ACG TTC ACC GAC TTA ACT CGC GAT GGTTGG CAC CAC ATG CGT GTA TTA CGC TTC CCG CCC CAA ACA TCC ACG CTG TCC CGTGGA ATT GGT GCG CAC ACT GAC TAT GGG TTG TTG GTA ATT GCC GCT CAG GAC GATGTT GGT GGC TTA TAT ATT CGC CCT CCA GTC GAG GGA GAG AAG CGT AAT CGT AACTGG TTG CCT GGT GAG AGC TCA GCA GGC ATG TTT GAG CAC GAT GAA CCT TGG ACCTTC GTG ACG CCC ACC CCA GGC GTG TGG ACA GTT TTC CCA GGT GAT ATC TTG CAGTTC ATG ACC GGC GGC CAG CTG CTT TCC ACT CCG CAC AAG GTT AAG CTC AAT ACCCGC GAA CGT TTC GCC TGC GCT TAT TTT CAT GAG CCT AAT TTT GAA GCA TCC GCCTAT CCG TTG TTC GAG CCC AGC GCC AAT GAG CGT ATT CAT TAT GGT GAG CAC TTTACC AAC ATG TTT ATG CGT TGC TAT CCA GAT CGG ATC ACC ACC CAG AGG ATC AACAAG GAG AAT CGC CTG GCG CAC TTG GAG GAC TTG AAG AAG TAT TCG GAC ACC CGCGCG ACA GGC TCA
 4. A DNA fragment according to claim 1 which comprises agene encoding an ethylene-forming enzyme of bacteria, wherein saidfragment has a base sequence of at least from No. 519 to No. 1568 of thefollowing base sequence:SEQ ID NO:3: ##STR1##
 5. A vector which includesa DNA fragment which encodes the amino acid sequence defined in SEQ IDNO:1.
 6. A host cell which has been transformed by the vector of claim5.
 7. The host cell of claim 6 wherein said host cell is E. coli.